Method For Measuring Protozoan Oocyst and Detecting Reagent

ABSTRACT

The present invention provides a method for measuring oocyst of protozoa, such as  Cryptosporidium , in an environment sample with high sensitivity at low cost within a short period of time; and a detecting reagent for use therein. 
     Magnetic fine particles of 5 to 500 nm particle diameter having, immobilized thereto, binding factors for specific recognition of oocyst are added to an analyte containing a protozoan oocyst to form oocyst/binding factor/magnetic fine particle complexes by using a binding reaction to the oocyst, the formed complexes are recovered by a magnetic separation, and the protozoan oocysts contained in the complexes are assayed. Further, there is provided, for conducting the above method, a reagent for detecting protozoan oocysts comprising magnetic fine particles of 5 to 500 nm particle diameter having, immobilized thereto, antibodies against oocysts or binding factors for recognizing the antibodies.

TECHNICAL FIELD

The present invention relates to a method for measuring protozoan oocystand a reagent for detecting the same, capable of inexpensively andconveniently detecting the presence of a protozoan oocyst in variousenvironments such as raw water for tap water, waste water, sewage,natural water and soil.

BACKGROUND ART

Tap water, groundwater, stream water, and the like are ingested asdrinking water and also there is a possibility that they areunconsciously taken up from mouth, so that it is necessary to payattention to their treatment in view of good hygiene. In addition tobacteria and suspended particulates, attention has been recently paid toprotozoa such as Cryptosporidium.

Cryptosporidium is a digestive tract-parasitic protozoan which isparasitic on mucous membranes of stomach and intestinal tracts to causediarrhea. Cryptosporidium proliferates with repeating an asexualreproduction term and a sexual reproduction term and oocysts generatedfrom the result of sexual reproduction are discharged into feces ofparasitic hosts. Since the oocyst is stable and maintains activity for along period of time, contamination of stream water and groundwater withoocysts discharged into for some reasons into drinking water may invitea situation that the oocyst infects human. In an infection example whichoccurred in Ogose-cho, Saitama-prefecture in 1996, a sewage-treatmentplant exists at the upper stream of a river of raw water for tap waterand thus oocyst as a primary infection source is considered to enterinto tap water through a drinking-water treatment plant using the streamwater as water source. Furthermore, discharged water from lavatoriesused by patients developing symptoms had been treated as sewage andagain flew into the river and the stream water was utilized as a watersource for tap water, so that infection was extended successively andfinally a half of citizens were infected.

Oocyst of Cryptosporidium has an extremely strong resistance againstdisinfection by chlorine and treatment with ozone and thus it isimpossible to completely annihilate the oocysts in water by usualwater-purifying treatment. Therefore, in order to prevent infection withCryptosporidium via water, it is necessary to assay a minute amount ofoocysts in a sample in high accuracy together with sufficient removal ordisinfection of the pathogenic protozoan.

Ministry of Health, Labor and Welfare has determined a guideline fortentative measure on preventive action and emergency action againstthese chlorine-resistant microorganisms (e.g., see Non-Patent Document1). In the document, various methods for measuring Cryptosporidium arelisted, including descriptions of an operating method comprising threesteps: a “concentration step” where a collected sample is concentratedby one of the methods such as suction filtration, pressure filtration,cartridge filter method, or centrifugal precipitation, a“separation/purification step” where protozoan oocysts are separatedfrom other suspending substances and purified by a method such asdensity-gradient centrifugal precipitation or immunological magneticparticle method (immunological magnetic bead method), and a subsequent“staining/microscopic inspection step” where the protozoan oocysts areimmunologically stained and measured on a microscope; or an operatingmethod comprising the above “concentration step” and“staining/microscopic inspection step”.

However, in the case that oocysts are separated from an environmentalsample using a centrifugal means, specific gravity of Cryptosporidium isclose to specific gravities of water and other contaminants and henceoocysts of Cryptosporidium present in the environmental sample cannot becompletely recovered by conducting common low-speed centrifugalseparation alone.

Moreover, in the guideline for tentative measure of Ministry of Health,Labor and Welfare, an immunological magnetic bead method is recommendedas the above “separation/purification step” in the detection andmeasurement of oocysts of Cryptosporidium. This method is a methodwherein immunological magnetic beads of 5 to 6 μm diameter are added toa sample subjected to the concentration step to effect an immunereaction and the oocysts bound onto the beads are magnetically recoveredtogether with the beads. It may be convenient to observe the recoveredoocysts directly through immunological fluorescent staining butactually, it is impossible to discriminate the oocysts because theimmunological magnetic beads exhibit autofluorescence and also resemblethe oocysts in size. Therefore, there is required a step of dissociatingthe oocysts from the magnetic beads having the oocysts bound theretowith hydrochloric acid. After the dissociation, a portion of the aciddissociation liquid containing the oocysts is placed on a glass slideand then neutralized with an alkali. After the solution of thepreparation is air-dried, it is washed with methanol and then theoocysts are stained with fluorescent antibodies. The staining takes 30minutes. Furthermore, in order to remove unbound fluorescent antibodies,washing is conducted but full attention should be paid so as not to washout the oocysts from the preparation. Moreover, since ahigh-concentration salt is precipitated by the neutralization, thestaining is not homogeneously effected and it is difficult todiscriminate fluorescent oocysts from background in some cases. Also, inthe operation for dissociation, all the oocysts are not alwaysdissociated and some oocysts may remain on the magnetic beads, so thatthe recovery ratio is also problematic. Thus, the conventional methodwith the micron-size magnetic beads has defects that the steps aretedious and complex, a lot of skills are required for fluorescentstaining, and also the recovery ratio of oocysts is insufficient.

Particularly, in the assay of tap water, since it is necessary to findone or two oocysts in a large amount of water, it is difficult even forthose who attend skill-training to decide whether a substance emittingfluorescence belongs to Cryptosporidium or not and there are evenconfused cases induced by reports of mistaken detection, so that variousproblems remain on the method for detecting Cryptosporidium and thuswater quality criteria therefor has not yet been defined. The reasonsfor requiring considerable skill in this assay method are that it takesa lot of time to concentrate Cryptosporidium from an analyte and that itrequires the skill to discriminate Cryptosporidium oocysts under afluorescent microscope.

As another staining/microscopic inspection step, anti-acid staining hasbeen also developed but non-specific reaction with substances other thanoocysts occurs remarkably in this method and thus there is a problemthat the judgment of oocyst is difficult for a person who is notconsiderably skilled specialist.

As the other method for detecting Cryptosporidium, there has been knowna method for detecting a specific DNA sequence of Cryptosporidium usinga PCR process (e.g., see Patent Document 1). The method is a methodwherein Cryptosporidium recovered by centrifugation is treated accordingto conventional procedures such as proteolytic treatment usingproteinase K, phenol-chloroform treatment, and ethanol precipitation torecover DNA and then a sequence specific to Cryptosporidium is amplifiedby a PCR process to detect the presence of Cryptosporidium.

However, this method employs a thermal cycler for amplifying DNA and aprimer having a specific base sequence and hence is by no means aninexpensive and convenient method.

Furthermore, in both of the method using a specific antibody and amethod of recognizing a specific DNA sequence by a PCR process,centrifugation is used at the time when Cryptosporidium is recovered.However, since specific gravities of oocysts and sporozoites ofCryptosporidium are near to 1, there is a large loss part impossible torecover by general low-speed centrifugation and hence there is a problemthat Cryptosporidium present in a sample cannot be sufficientlydetected.

-   Non-Patent Document 1: Measure against Cryptosporidium in Tap Water,    supervised by Ministry of Health and Welfare, Life Hygiene Bureau,    Water Environment Division, Water Maintenance Department, published    by K. K. Gyousei (December, 1999), pp. 1-2.-   Patent Document 1: JP-A-11-243953

DISCLOSURE OF THE INVENTION

Problems that the Invention is to Solve

Accordingly, an object of the invention is to provide a method ofmeasuring oocyst of protozoa, such as Cryptosporidium, in an environmentsample with high sensitivity at low cost within a short period of time.The invention provides a convenient and highly sensitive method formeasuring protozoan oocyst and a detecting reagent for use therein.

Means for Solving the Problems

As a result of the extensive studies, the present inventors have foundthat the above problems are solved by the use of magnetic fine particlesof 5 to 500 nm particle diameter (hereinafter also referred to as“magnetic nanoparticle”), which are different from micron-size magneticbeads for use in the conventional immunological magnetic beads method,and thus have accomplished the invention based on the findings.

Namely, the above object can be achieved by the following constitutions.

(1) A method for measuring protozoan oocysts, which comprises:

adding magnetic fine particles of 5 to 500 nm particle diameter, whichhave a binding factor for specifically recognizing protozoan oocystsimmobilized thereto, to an analyte containing protozoan oocysts to formcomplexes of the oocysts with the magnetic fine particles through thebinding factor;

recovering the thus formed oocyst/binding factor/magnetic fine particlecomplexes by a magnetic separation; and

counting the number of oocysts.

(2) The method for measuring protozoan oocysts according to the above(1), wherein the binding factor is an antibody against oocyst(hereinafter referred to as “antioocyst antibody”).

(3) The method for measuring protozoan oocysts according to the above(1) or (2), wherein the oocyst/binding factor/magnetic fine particlecomplexes are oocyst/antioocyst antibody/magnetic fine particlecomplexes formed by adding magnetic fine particles having the antioocystantibody immobilized thereto to the analyte.

(4) The method for measuring protozoan oocysts according to the above(1), wherein the binding factor comprises the antioocyst antibody and abinding factor component specifically recognizing the antibody(hereinafter referred to as “antioocyst antibody-binding factorcomponent”).

(5) The method for measuring protozoan oocysts according to the above(1), wherein the oocyst/binding factor/magnetic fine particle complexesare oocyst/antioocyst antibody/antioocyst antibody-binding factorcomponent/magnetic fine particle complexes, which are formed by addingantibodies against oocyst to an analyte to form oocyst/antioocystantibody complexes, and subsequently adding magnetic fine particleshaving the antioocyst antibody-binding factor component against theantibody immobilized thereto.

(6) The method for measuring protozoan oocysts according to any one ofthe above (1) to (5), wherein the magnetic fine particles are labeledbeforehand.

(7) The method for measuring protozoan oocysts according to any one ofthe above (1) to (5), wherein the formed oocyst/binding factor/magneticfine particle complex is further labeled.

(8) The method for measuring protozoan oocysts according to the above(6) or (7), wherein the labeling is a fluorescent labeling.

(9) The method for measuring protozoan oocysts according to any one ofthe above (1) to (8), wherein the magnetic fine particles are magneticfine particles having stimuli-responsive polymers immobilized thereto.

(10) The method for measuring protozoan oocysts according to any one ofthe above (1) to (9), wherein the protozoan is a protozoan belonging togenus Cryptosporidium.

(11) The method for measuring protozoan oocysts according to any one ofthe above (1) to (10), wherein the analyte contains water as a solvent.

(12) A reagent for detecting protozoan oocysts in an analyte, whichcomprises magnetic fine particles of 5 to 500 nm particle diameterhaving antioocyst antibodies or antioocyst antibody-binding factorcomponents immobilized thereto.

(13) The reagent for detecting protozoan oocysts according to the above(12), wherein the above magnetic fine particles are magnetic fineparticles having stimuli-responsive polymers immobilized thereto.

ADVANTAGE OF THE INVENTION

According to the invention, there can be obtained a method for measuringprotozoan oocyst and a reagent for detecting protozoan oocyst, whichallows detection of protozoan oocyst conveniently and rapidly in highaccuracy without requiring skilled specialists.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1]

FIG. 1 is a schematic drawing of an immunocomplex. FIG. 1A is aschematic drawing of an immunocomplex (Cryptosporidiumoocyst/antiCryptosporidium oocyst antibody/magnetic bead complex) usinga conventional magnetic bead method. FIG. 1B is a schematic drawing of aCryptosporidium oocyst/antiCryptosporidium oocystantibody/antiCryptosporidium oocyst antibody binding factorcomponent/stimuli-responsive polymer/magnetic nanoparticle complex usingmagnetic nanoparticles according to the invention.

[FIG. 2]

FIG. 2A is an immunological fluorescent microscopic photograph ofCryptosporidium oocysts separated with UCST-type heat responsivemagnetic nanoparticles and FIG. 2B is an immunological fluorescentmicroscopic photograph of Cryptosporidium oocysts in an aggregated stateseparated with UCST-type heat responsive magnetic nanoparticles. FIG. 2Cand FIG. 2D are bright-field microscopic photographs of FIG. 2A and FIG.2B, respectively.

[FIG. 3]

FIG. 3A and FIG. 3C are bright-field microscopic photographs ofcommercially available magnetic beads and FIG. 3B and FIG. 3D arefluorescent microscopic photographs of FIG. 3A and FIG. 3C,respectively.

[FIG. 4]

FIG. 4A and FIG. 4B are bright-field microscope photographs of yeastcells and oocysts before immunological fluorescent labeling. FIG. 4C andFIG. 4D are a bright-field microscope photograph and a fluorescentmicroscopic photograph of the yeast cells after immunologicalfluorescent labeling, respectively.

FIG. 4E and FIG. 4F are a bright-field microscope photograph and afluorescent microscopic photograph of an excess amount of the yeastcells and Cryptosporidium oocysts subjected to immunological fluorescentlabeling after they are mixed, respectively.

[FIG. 5]

FIG. 5A is a bright-field microscopic photograph of magneticnanoparticle aggregates separated with UCST-type thermo-responsivemagnetic nanoparticles and FIG. 5B is a fluorescent microscopicphotograph thereof. FIG. 5C is a bright-field microscopic photograph ofremaining supernatant after separated with UCST-type thermo-responsivemagnetic nanoparticles and FIG. 5D is a fluorescent microscopicphotograph thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

In the invention, to an analyte containing protozoan oocyst is addedmagnetic fine particles of 5 to 500 nm particle diameter having,immobilized thereto, binding factors for specific recognition of theoocysts, thereby oocyst/binding factor/magnetic nanoparticle complexesbeing formed. Thus, owing to the fine particle property, the bindingreaction with the oocyst extremely rapidly proceeds and also noautofluorescence is observed on the magnetic nanoparticles, so thatthere becomes unnecessary a step of dissociating magnetic particle fromthe oocysts with hydrochloric acid which is conventionally an essentialstep. Namely, the complex can be recovered and subjected to the nextdetection step in the state of the “oocyst/binding factor/magneticnanoparticle complex” wherein magnetic nanoparticles and oocysts arebound through the binding factor, and hence rapidness and recovery ratioare remarkably improved.

FIG. 1A schematically represents a Cryptosporidiumoocyst/antiCryptosporidium oocyst antibody/magnetic bead complex bymeans of an immunological magnetic beads having, immobilized thereto, anantibody against oocyst of protozoa belonging to Genus Cryptosporidium(hereinafter referred to as “antiCryptosporidium oocyst antibody”).

FIG. 1B schematically represents a complex of magnetic nanoparticlehaving, immobilized thereto, a stimuli-responsive polymer bound to ananticryptosporidium oocyst antibody binding factor component with ananticryptosporidium oocyst antibody-Cryptosporidium oocyst complex.

The conventional immunological micron-size magnetic beads haveaforementioned defects. To the contrary, with regard to the magneticnanoparticles for use in the invention, sufficient dispersibility isobtained owing to the remarkably small particle diameter and hence therecovery ratio of oocysts existing in a minute amount is improved. Theimprovement of dispersibility results in a rapid binding reaction, whichleads to shortening of the step. One hour of the reaction time requiredin the case of the conventional micron-size magnetic beads is shortenedto several tens seconds in the case of the magnetic nanoparticles foruse in the invention. Particularly, in the magnetic nanoparticleshaving, immobilized thereto, the stimuli-responsive polymers describedin FIG. 1B, since the aggregate is formed depending on change intemperature or pH, it is easy to recover the dispersed magnetic fineparticles. Namely, when the dispersed magnetic nanoparticles areaggregated by changing temperature or pH, the particles can be easilyrecovered by setting a magnetic plate or the like. Therefore,centrifugation or the like is not necessary. In addition, since themagnetic nanoparticles and the stimuli-responsive polymers exhibit noautofluorescence, it is possible to enter the next detection(labeling/microscopic inspection) step after the recovering operationwithout separating the magnetic beads from oocysts. The easiness ofdispersion/recovery simplifies the staining step. Specifically, a buffersolution and fluorescent antibodies are added to the recovered magneticnanoparticles and pipetting is continued in a dispersed state for dozensof seconds. Then, the particles are converted into an aggregated stateand recovered with a magnetic plate, and then the resulting supernatantis discarded. A buffer solution is further added thereto and pipettingis further continued in a dispersed state for dozens of seconds. Then,the particles are converted into an aggregated state and recovered witha magnetic plate, and then the resulting supernatant is discarded,followed by washing. The operations are further repeated twice to obtaina highly purified sample for observation.

Moreover, in the invention, a binding method for formation of thecomplex of the magnetic nanoparticles with oocysts, a timing and sitefor binding them are arbitrary. For example, immunocomplexes may beformed by preparing magnetic nanoparticles having an antibody againstoocyst immobilized thereto beforehand and adding the particles to ananalyte containing oocysts. Alternatively, an oocyst/antioocystantibody/antioocyst antibody binding factor component/magnetic fineparticle complexes may be formed by adding an antibody against oocyst toan analyte containing oocysts to form an oocyst/antioocyst antibodycomplex and adding magnetic nanoparticles having, immobilized thereto, abinding factor recognizing the antioocyst antibody into the sample.Also, biotin, avidin, a biotinylated antibody, and the like can befreely utilized. A complex may be directly formed between antioocystantibody and magnetic nanoparticles or a complex may be formedindirectly through biotin, avidin, biotinylated antibody, or the like.

Moreover, in the invention, a complex labeled in one step can be formedby forming a complex with oocyst using magnetic nanoparticles labeledbeforehand. In this case, also, shortening of the step and improvementof accuracy are achieved.

Furthermore, when magnetic nanoparticles having, immobilized thereto,stimuli-responsive polymers containing a binding factor bound theretoare used as magnetic nanoparticles, the oocysttimuli-responsive polymerimmobilized magnetic nanoparticle complex is easily aggregated byimparting the corresponding stimulation, so that recovery of the complexis facilitated and thus the case is preferable.

The following will describe the invention further in detail.

First, there is described an antibody against protozoan oocyst ormagnetic nanoparticle having, immobilized thereto, a binding factorcomponent specifically recognizing the antibody.

The material for the magnetic nanoparticles for use in the invention maybe an organic substance or inorganic substance so far as it is asubstance exhibiting magnetism at ordinary temperature. The substanceexhibiting magnetism is not particularly limited but specificallyincludes nickel oxide particles, ferrite particles, magnetite particles,maghemite particles, cobalt iron oxide, barium ferrite, carbon steel,tungsten steel, KS steel, particles of rare-earth cobalt magnets,hematite particles, and the like.

The particle diameter of the magnetic nanoparticles is in the range of 5to 500 nm, preferably in the range of 20 to 150 nm. When the particlediameter of the magnetic nanoparticles is adjusted to this range,surface area per unit volume of the magnetic nanoparticles can beincreased and hence sensitivity toward an antigen (oocyst) in animmunological magnetic bead method can be remarkably improved. Moreover,shortening of the time for the binding reaction can be also achieved.When the particle size of the magnetic nanoparticles is less than 5 nm,the time for magnetic separation is necessary and it requires a lot oftime. When the size exceeds 500 nm, there is a risk of decrease inrecognizing ability toward oocyst.

The method for preparing the magnetic nanoparticle for use in theinvention is described with reference to the case using magnetite as anexample. Magnetic magnetite fine particles having a particle diameter ofseveral tens nm can be obtained by converting magnetite into doublemicelles using sodium oleate and sodium dodecylbenzenesulfonate anddispersing the micelles into an aqueous solution. This method is amethod described in Biocatalysis, 1991, Vol. 5, pp. 61-69.

The binding factor to be immobilized to the magnetic nanoparticles isnot particularly limited so far as it is a molecule having a specificbinding ability toward protozoan oocyst and having a function capable ofbinding oocyst to the magnetic nanoparticles to form a complex. Thefactor may be constituted by a single component or may be constituted byplurality of binding factor components. Moreover, an antibody or thelike may be used as the binding factor. In the invention the complex ofmagnetic nanoparticles containing an antibody such as antioocystantibody is sometimes referred to as an immunological magneticnanoparticles, and the complex of oocysts and the immunological magneticnanoparticles is sometimes referred to as an immunocomplex.

Moreover, the method for immobilization thereof is not particularlyrestrictive and conventionally known methods may be used. As the methodfor immobilizing the binding factor on the magnetic nanoparticles, inthe case of using an antibody, for example, there may be mentioned amethod of immobilizing the antibody onto the nanoparticle surface by acondensation or addition reaction with a functional group (e.g.,carboxyl group, amino group, or epoxy group) immobilized onto thesurface of the nanoparticles beforehand utilizing an amino acid residue(e.g., amino group, carboxyl group, or the like) existing on the surfaceof the binding factor.

Furthermore, as a biochemical procedure, there may be mentioned a methodof immobilizing biotin to the surface layer of the nanoparticlesbeforehand, binding avidin having a specific binding ability towardbiotin, further forming a biotinylated antibody using a commerciallyavailable biotinylation kit, and subsequently immobilizing an antibodyonto the surface of the nanoparticles utilizing an avidin-biotin bondformed in an aqueous solution.

As specific forms mediating the bond between the magnetic nanoparticlesand oocysts, there may be, for example, mentioned biotin and avidin, anantigen and an antibody, a polynucleotide and a polynucleotide having abase sequence complementary to the polynucleotide, an enzyme (activesite) and a substrate, an enzyme (active site) and a product, an enzyme(active site) and a competitive inhibitor, an enzyme (coenzyme bindingsite) and a coenzyme, an enzyme (coenzyme binding site) and a triazinepigment, an Fc site and protein A, an Fc site and protein G, lectin anda sugar, a hormone receptor and a hormone, a DNA and a DNA bindingprotein, heparin and fibronectin, and heparin and laminin.

In the invention, as the magnetic nanoparticles, it is preferred to usethose having a stimuli-responsive polymer immobilized thereto. Thereby,the magnetic nanoparticles having the above stimuli-responsive polymersimmobilized thereto can be precipitated and aggregated by imparting thecorresponding stimulation and thus recovery of the magneticnanoparticles by means of a magnet can be more efficiently conducted.

The stimuli-responsive polymer herein means a polymer having a propertyof precipitating and aggregating the polymer in a solvent containing thepolymer in response to stimulation such as temperature, pH, light,magnetic field, or electricity. When the above stimuli-responsivepolymer is immobilized to the magnetic nanoparticle, after oocyst andimmunological magnetic nanoparticle form an immunocomplex, theimmunocomplex is aggregated by imparting stimulation to the solventcontaining the immunocomplex, so that it can be easily magneticallyseparated (recovered). Any of the conventionally knownstimuli-responsive polymers can be used.

In the invention, as preferred stimuli-responsive polymers, there maybe, for example, mentioned a pH responsive polymer wherein a physicalproperty (solubility toward a solvent, a form, or the like) responds tochange in pH, a light responsive polymer wherein a physical propertyresponds to change in wavelength of light, and a heat responsive polymerwherein a physical property responds to change in temperature.Particularly preferred is a heat responsive polymer. The heat responsivepolymer means a polymer which reversibly repeats aggregation anddissolution in an aqueous solution upon temperature change. As the heatresponsive polymer, there are known a polymer having lower criticalsolution temperature (LCST) and a polymer having upper critical solutiontemperature (UCST). Specifically, poly-N-isopropylacrylamide is known asa polymer exhibiting LCST in an aqueous solution and its phasetransition temperature is 32° C. A copolymer of acrylamide andN-acetylacrylamide or the like is known as a polymer exhibiting UCST inan aqueous solution and its phase transition temperature can be varieddepending on the ratios of individual monomer components.

As the heat responsive magnetic nanoparticles, there may be mentionedmagnetic particles described in International Publication WO02/16528pamphlet, International Publication WO02/16571 pamphlet, and the like.

The following will describe the measuring method of the invention.

In the invention, the “environmental sample” means raw water for tapwater, soil, waste water, sewage, pool water, feces, or the like. Theenvironmental sample may be subjected to a concentration step accordingto need and thus may be the analyte for subjecting to thepurification/separation step of the invention.

In the invention, the “oocyst” means one wherein zygote is enwrappedwith a membrane. The zygote is divided inside the oocyst to formsporozoite having infectivity. An oocyst has a characteristic that it isextremely resistant to environmental change, such as dryness andchemicals, owing to the presence of the shell.

In the invention, in the recovery of protozoan oocyst by theconventional immunological magnetic bead method, it becomes possible torecover protozoan oocyst in higher sensitivity by the use of magneticnanoparticles instead of the conventional micron-size magnetic beads.

For example, in the case of using magnetic nanoparticles having UCSTpolymers bound thereto, by adding into an analyst the magneticnanoparticles having an antibody against oocyst immobilized thereto andstirring the whole with pipetting operation or the like for dozens ofseconds (preferably 20 to 60 seconds), it is possible to form an“immunocomplex” wherein oocysts and magnetic nanoparticles are boundthrough the antibody. As compared with the reaction time required in theconventional micron-size immunological magnetic bead method. i.e., 1hour, the reaction time can be remarkably shortened.

Thereafter, the immunocomplex is recovered by magnetic separation. Asthe magnetic separation method, conventionally known methods can beemployed.

The magnetic nanoparticles having stimuli-responsive polymers accordingto the invention immobilized thereto repeat aggregation/dissolution uponchange in heat, thermal shock, or pH. Therefore, when the above magneticnanoparticles added to the reaction solution are aggregated beforehandby change in heat or pH, the particles can be easily separated with amagnet. Owing to small particle diameter, reactivity for recognitionbinding is high and separation is easy, so that a magnetic column or thelike is not necessary. Therefore, the stimuli-responsivepolymer-immobilized magnetic fine particles (stimuli-responsive magneticnanoparticle) of 5 to 500 nm particle diameter having an antibodyagainst protozoan oocyst immobilized thereto are effective as adetecting reagent.

In the invention, particularly, by the use of heat responsive magneticnanoparticles which reversibly repeat dissolution/aggregation in anaqueous solution upon slight temperature change, Cryptosporidium oocystsin an environmental sample as an analyte can be efficiently recovered.This is because the above magnetic nanoparticles show excellentdispersibility in an appropriate temperature range. At the same time,since the above heat responsive magnetic nanoparticles are aggregatedupon temperature change, recovery thereof is easy.

For example, in the case of heat responsive magnetic nanoparticlesobtained by immobilization of an LCST polymer, the heat responsivemagnetic nanoparticles can be aggregated with heating operation. To thecontrary, in the case of heat responsive magnetic nanoparticles obtainedby immobilization of UCST polymers, the heat responsive magneticnanoparticles can be aggregated with cooling operation. A washingoperation is facilitated by magnetic separation of the aggregate throughprompt action of a magnet and discarding supernatant. Furthermore, theaggregate can be sufficiently dispersed with cooling in the case of heatresponsive magnetic nanoparticles obtained by immobilization of LCSTpolymers and with heating in the case of heat responsive magneticnanoparticles obtained by immobilization of UCST polymers, so thatremoval of non-specifically bound matter and removal of unbound antibodyfor fluorescent staining can be efficiently conducted. Each of the stepsfor dispersion/aggregation takes only dozens of seconds.

Then, the number of the oocyst in the analyte is measured by conductinga detecting step of the oocyst. In the invention, the oocyst to whichthe above magnetic nanoparticles are bound can be subjected to the nextdetection step as they are (without elution/dissociation of the magneticfine particles).

In the invention, the above immunocomplex is preferably labeled by aconventionally known method and thereby, the oocyst can be easilydetected by microscopic inspection. Moreover, in the invention, asmentioned above, since it is not necessary to dissociate magneticnanoparticles, a “labeled complex” can be obtained in one stage by theuse of magnetic nanoparticles labeled beforehand.

The detection step using a label usable in the invention is notparticularly limited so far as it includes a step of labeling andmeasuring a complex formed by reacting the oocyst and the magneticnanoparticles and a method usually used in the art can be suitablyapplied. For example, any of a direct method, an indirect method, ahomogeneous method, a heterogeneous method, and the like in immunoassaymay be used. Moreover, there may be employed any of radioimmunoassayusing RI, enzyme immunoassay using an enzyme such as alkalinephosphatase or peroxidase, fluorescent immunoassay using a fluorescentsubstance, as the label for use in detection of the complex.

Preferred is a method of labeling with a fluorescent substance andfluorescent substances commonly used in the immunofluorescence methodmay be suitably used. For example, there may be mentioned fluorescein,rhodamine, sulforhodamine 101, lucifer yellow, acridine, riboflavin, andthe like. Preferred is fluorescein.

It is desired that the number of the oocysts of Cryptosporidium existingin the environmental sample is detected at a unit of several oocysts.Drinking water such as raw water for tap water should not becontaminated with any oocyst of Cryptosporidium. In an environmentalsample, particularly a sample relating to tap water or drinking water,it is required to detect only 1 or several oocysts. In the aboveobservation method, it is possible to correctly and clearly count thenumber of oocyst of Cryptosporidium existing in the environmentalsample.

The invention will be further described in the following Examples butthe description explains the invention further in detail and does notlimit the scope of the invention.

EXAMPLE 1

Detection of Cryptosporidium Oocyst Using UCST-Type Thermo-ResponsiveMagnetic Nanoparticles

(i) Preparation of Standard Solution of Cryptosporidium Oocyst

A 500 μl portion of a commercially available standard reagent ofCryptosporidium parvum oocyst (Waterborne™, Inc., 1.25×10⁶/ml) wasplaced in a 1.5 ml tube, followed by centrifugation (10000 rpm) at roomtemperature for 5 minutes. After removal of the supernatant undersuction, 0.5 ml of TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% bymass Tween 20, pH 7.5) was added to the tube and the whole was subjectedto vortex stirring. Furthermore, a commercially availableimmunofluorescent labeling reagent FITC-Anti-Cryptosporidium IgM (mAb)(Waterborne™, Inc., A400FLR-20X) was added in an amount which is 1/2000of the liquid volume, followed by a reaction at room temperature for 10minutes. Thereafter, 2 μl (KPL 0.5 ug/μl) of a commercially availablebiotinylated antimouse anti-IgM secondary antibody (IgG), followed by areaction at room temperature for 10 minutes. Then, after 5 μl (10 mg/ml)of avidin was added thereto and the whole was allowed to stand at roomtemperature for 10 minutes, centrifugation (10000 rpm at roomtemperature) was conducted for 5 minutes and the supernatant wasremoved. Then, 0.5 ml of TBST buffer was added and unreacted FITC andanti-Cryptosporidium primary antibody, anti-IgM secondary antibody, andavidin were removed. A complex, named Cryptosporidiumoocyst/antiCryptosporidium oocyst fluorescent antibody/antifluorescentantibody biotinylated antibody/avidin was obtained, which was labeledwith fluorescence and was bound to a biotinylated antibody through afluorescent antibody and to avidin through biotin.

(ii) Separation of Cryptosporidium Oocyst

To 500 μl of an aqueous solution of Cryptosporidiumoocyst/antiCryptosporidium oocyst fluorescent antibody/antifluorescentantibody biotinylated antibody/avidin prepared by the above operationswas added 50 μl of 1% by mass UCST-type biotinylated thermo-responsivemagnetic nanoparticles. After standing at 42° C. for 2 minutes, a testtube was set on a magnet stand and was immersed and cooled in an icebath at 0° C. for 5 minutes to conduct magnetic recovery ofCryptosporidium oocysts.

After magnetic separation, the supernatant was removed under suction bymeans of a pipette, 0.5 ml of TBST buffer was again added, and theUCST-type thermo-responsive magnetic nanoparticles were again dispersedat room temperature. After standing for 2 minutes, the UCST-typethermo-responsive magnetic nanoparticles dispersed were again aggregatedby re-immersion into an ice bath and magnetic recovery of the UCST-typethermo-responsive magnetic nanoparticles by a magnet was conducted. Theoperations were further repeated twice and finally, 50 μl of TBST bufferwas added to the aggregate of the UCST-type thermo-responsive magneticnanoparticles from which washing liquid had been removed. The UCST-typethermo-responsive magnetic nanoparticles were again suspended with apipette to prepare a sample for analyte.

(iii) Detection of Cryptosporidium Oocyst

A 2 μl portion of the sample for analyst thus prepared was dropped on aglass slide for observation without eluting the Cryptosporidium oocystsfrom UCST-type thermo-responsive magnetic nanoparticles, to form apreparation for observation.

As a result of observation of the above preparation, Cryptosporidiumoocysts were efficiently detected as shown in FIG. 2.

Namely, FIG. 2A is an immunological fluorescent microscopic photographof Cryptosporidium oocysts separated with the UCST-typethermo-responsive magnetic nanoparticles and FIG. 2C is a bright-fieldmicroscopic photograph thereof. In FIG. 2A, a fluorescent image of theUCST-type thermo-responsive magnetic nanoparticles is not observed andthus it is found that an efficient observation image is obtained withoutdissociation from the magnetic nanoparticles. Moreover, FIG. 2B is animmunological fluorescent microscopic photograph of Cryptosporidiumoocysts in an aggregated state separated with the UCST-typethermo-responsive magnetic nanoparticles. The arrow part in FIG. 2B isan aggregate part of the UCST-type thermo-responsive magneticnanoparticles but no fluorescent image is observed. The fact thatfluorescence is not observed even in the aggregated state clearlyindicates that it does not inhibit observation of the abovethermo-responsive magnetic nanoparticles. FIG. 2D is a bright-fieldmicroscopic photograph of FIG. 2B and the arrow part in FIG. 2D is anaggregate of the UCST-type thermo-responsive magnetic nanoparticles.

Thus, since no UCST-type thermo-responsive magnetic nanoparticles wereobserved in the observed fluorescent image of the Cryptosporidiumoocysts, it becomes apparent that separation of the UCST-typethermo-responsive magnetic nanoparticles from Cryptosporidium oocystswas not necessary after magnetic separation.

Furthermore, the supernatant after magnetic recovery was centrifuged(room temperature, 10000 rpm, 5 min) and recovery of unrecoveredCryptosporidium oocysts was attempted. After centrifugation, thesupernatant was gently sucked and discarded, 50 μl of a fresh TBSTbuffer solution was added to the tube, and fluorescent detection of theoocyst was conducted but fluorescent Cryptosporidium oocyst was notobserved in the supernatant. From the result, it was confirmed thatCryptosporidium oocysts were recovered from the analyte at an efficiencyof almost 100%.

COMPARATIVE EXAMPLE 1

A separation/detection test of Cryptosporidium by conventionalmicron-size magnetic beads was attempted.

(i) Observation of Micron-Size Magnetic Particles (Hereinafter alsoReferred to as Magnetic Beads) on a Fluorescent Microscope

Before the separation test of Cryptosporidium oocysts, observation of acommercially available micron-size magnetic beads on a fluorescentmicroscope was conducted.

FIG. 3A is a bright-field microscopic photograph of commerciallyavailable magnetic beads and FIG. 3B is a fluorescent microscopicphotograph of FIG. 3A. The color tone of fluorescence is notdiscriminated from fluorescent Cryptosporidium oocysts stained with FITCand also they extremely resemble each other in size. From this result,it was indicated that observation on a fluorescent microscope wasdifficult without elution of Cryptosporidium oocysts from the magneticbeads after recovery of Cryptosporidium oocysts.

(ii) Detection of Cryptosporidium Oocysts with CryptosporidiumOocyst-Detecting Kit Using Conventional Micron-Size Magnetic Beads

A 500 μl portion of a commercially available standard reagent ofCryptosporidium parvum oocyst (Waterborne™, Inc., 1.25×10⁶/ml) wasplaced in a 1.5 ml tube, followed by centrifugation (10000 rpm) at roomtemperature for 5 minutes. After removal of the supernatant undersuction, 0.5 ml of TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% bymass Tween 20, pH 7.5) was added to the tube and the whole was subjectedto vortex stirring. Then, 5 μl of Dynabeads Anti-Cryptosporidium(VERITAS) was added, followed by gentle shaking for 1 hour. Aftershaking, a test tube was set on a magnet stand, the magnetic beads werecollected and the supernatant was discarded. The test tube was removedfrom the magnet stand and 500 μl of TBST buffer was again added thereto,followed by gentle stirring for 30 minutes. Then, the test tube was seton the magnet stand, the immunological magnetic beads were collected,and the supernatant was discarded. The operations were further repeatedtwice and the beads were suspended in 50 μl of TBST buffer to prepare asample for microscopic inspection.

FIG. 3C is a bright-field microscopic photograph and FIG. 3D is afluorescent microscopic photograph of FIG. 3C. As shown by arrows inFIG. 3C and FIG. 3D, both of Cryptosporidium oocysts and the magneticbeads were observed on the bright-field microscopic photograph whereasonly the magnetic beads were observed and Cryptosporidium oocyst werenot observed on the fluorescent microscopic photograph. Those shown byarrows are Cryptosporidium oocysts separated by the commerciallyavailable magnetic beads but since they are not labeled withimmunofluorescence, Cryptosporidium oocysts are not observed on afluorescent microscope and thus commercially available magnetic beadsare only observed.

Moreover, the magnetic particles and Cryptosporidium oocysts extremelyresembled each other in both of size and shape as apparent from thebright-field and dark-field (fluorescent image) microscopic photographsin FIG. 3, it was confirmed that detection of Cryptosporidium oocysts ona fluorescent microscope was extremely difficult without elutionoperation of Cryptosporidium oocysts from the magnetic beads.

EXAMPLE 2 Comparison of Separation Efficiency Between UCST-TypeThermo-Responsive Magnetic Nanoparticles and Conventional Micron-SizeMagnetic Beads (i) Preparation of Sample

After 1.68×10, 1.68×10², 1.68×10³ or 1.68×10⁴ oocysts of a commerciallyavailable standard reagent of Cryptosporidium parvum oocysts(Waterborne™, Inc., 1.25×10⁶/ml) in 500 μl of TBST buffer was added to atube, a Cryptosporidium oocyst/antiCryptosporidium oocyst fluorescentantibody/antifluorescent antibody biotinylated antibody/avidin complexwas formed according to Example 1. Thereafter, in accordance withExample 1, the above complex was reacted with biotinylated UCST-typethermo-responsive magnetic nanoparticles to separate Cryptosporidiumoocysts. A finally recovered substance was suspended into 50 μl of aTBST buffer solution and the number of Cryptosporidium oocysts recoveredwas investigated on each sample.

(ii) Detection of Cryptosporidium Oocysts

In all the four samples, the equal number of oocysts to the number ofthe oocysts added in 500 μl of the TBST buffer was counted.

Moreover, the supernatant after magnetic recovery was furthercentrifuged (room temperature, 10000 rpm, 5 min) and separation ofCryptosporidium oocyst which had not been recovered was tried but thepresence was not confirmed.

COMPARATIVE EXAMPLE 2

An experiment similar to Example 2 was attempted using a commerciallyavailable Cryptosporidium oocyst-detecting kit. After 1.68×10, 1.68×10²,1.68×10³ or 1.68×10⁴ oocysts of a commercially available standardreagent of Cryptosporidium parvum oocysts (Waterborne™, Inc.,1.25×10⁶/ml) in 500 μl of TBST buffer was added to a test tube, 5 μl ofDynabeads Anti-Cryptosporidium (VERITAS) was added. After gentle shakingfor 1 hour, the test tube was set on a magnet stand and theimmunological magnetic beads were recovered and the supernatant wasdiscarded. The magnetic beads were washed by repeating the followingoperations: the test tube was removed from the magnet stand and 500 μlof TBST buffer was once again added thereto to disperse the magneticbeads, and further the magnetic stand was set to recover magnetic beadsand a supernatant was discarded. Then, 50 μl of 0.1N hydrochloric acidwas added to the recovered magnetic beads to dissociate Cryptosporidiumoocysts from the magnetic beads. A 50 μl portion of the dissociatedliquid was placed on a glass slide and neutralized by adding 5 μl of 1Nsodium hydroxide. After air-drying of the neutralized solution, 50 μl ofmethanol was added and the whole was again air-dried. To the air-driedsample was added 50 μl of a commercially available immunofluorescentlabeling reagent FITC-Anti-Cryptosporidium IgM (mAb) (Waterborne™, Inc.,A400FLR-20X) diluted to 1/2000, thereby fluorescent staining wasconducted. After staining, the fluorescent labeling antibody solutionwas removed and 50 μl of ion-exchanged water was further added andremoved to wash the sample, which was air-dried to form a preparationfor observation.

When the number of Cryptosporidium oocysts was investigated, fluorescentcounting results were lower than the theoretical number ofCryptosporidium oocysts for all the four samples.

Furthermore, when hydrochloric acid was again added to the magneticbeads, which had been magnetically recovered and from whichCryptosporidium had been dissociated, and detection of Cryptosporidiumoocysts was tried in a similar manner, fluorescent Cryptosporidiumoocysts were confirmed from the magnetic beads of all the four samples.The results indicate that recovery of Cryptosporidium oocyst is notcompleted by one dissociation operation. The observed counted numberlower than the theoretical one may be attributed to the low recoveryratio.

EXAMPLE 3

Separation of Cryptosporidium Oocysts From a Mixed Liquid with YeastCells with UCST-Type Thermo-Responsive Magnetic Nanoparticles

Fluorescent staining of Cryptosporidium oocysts was attempted in thepresence of an excess amount of yeast cells (Saccharomyces cerevisiae).Into 500 μl of TBST buffer were mixed 1×10⁶ yeast cells (see FIG. 4A,which is a bright-field microscope photograph of yeast cells beforeimmunofluorescent labeling) and 3×10⁵ Cryptosporidium oocysts (see FIG.4B, which is a bright-field microscope photograph of oocysts beforeimmunofluorescent labeling). Then, 50 μl of a commercially availableimmunofluorescent labeling reagent FITC-Anti-Cryptosporidium IgM (mAb)(Waterborne™, Inc., A400FLR-20X) diluted to 1/2000 was added, therebyfluorescent labeling was conducted. Centrifugation at room temperaturefor 1000 rpm for 5 minutes was conducted and then the precipitate wasrecovered and the supernatant was discarded. Further, TBST buffer wasadded and the whole was stirred, followed by centrifugation at roomtemperature for 1000 rpm for 5 minutes. After this operation was furtherrepeated twice, the precipitate was dispersed into 50 μl of TBST bufferto prepare a sample for observation.

As a result, as shown by arrows in FIG. 4C to FIG. 4F, in both ofimmunological fluorescent images of the yeast cells alone (see FIG. 4Cand FIG. 4D; FIG. 4C is a bright-field microscope photograph and FIG. 4Dis a fluorescent microscopic photograph of the yeast cells afterimmunofluorescent labeling) and the case where the yeast cells andCryptosporidium oocysts were mixed (see FIG. 4E and FIG. 4F; images ofan excess amount of the yeast and Cryptosporidium oocyst subjected toimmunofluorescent labeling after they are mixed; FIG. 4E is abright-field microscope photograph and FIG. 4F is a fluorescentmicroscopic photograph thereof), the arrowed yeast cells were notconfirmed on a fluorescent microscope and only the Cryptosporidiumoocysts were observed (see FIG. 4F).

Moreover, separation of Cryptosporidium oocysts from an analyte (liquid)containing the yeast cells and Cryptosporidium oocysts in the aboveconcentrations was conducted in accordance with Example 1.

As a result, only Cryptosporidium oocysts were detected from magneticaggregate of UCST-type thermo-responsive magnetic nanoparticles but noyeast cells were detected (see FIG. 5A and FIG. 5B; FIG. 5A is abright-field microscopic photograph of magnetic nanoparticle aggregatesseparated with UCST-type thermo-responsive magnetic nanoparticles andFIG. 5B is a fluorescent microscopic photograph thereof).

On the other hand, in the supernatant after magnetic recovery, only theyeast cells were detected but no Cryptosporidium oocysts were detected(see FIG. 5C and FIG. 5D; FIG. 5C is a bright-field microscopicphotograph of remaining supernatant after separated with UCST-typethermo-responsive magnetic nanoparticles and FIG. 5D is a fluorescentmicroscopic photograph thereof).

1. A method for measuring protozoan oocysts, which comprises: addingmagnetic fine particles of 5 to 500 nm particle diameter, which have abinding factor for specifically recognizing protozoan oocystsimmobilized thereto, to an analyte containing protozoan oocysts to formcomplexes of the oocysts with the magnetic fine particles through thebinding factor; recovering the thus formed oocyst/bindingfactor/magnetic fine particle complexes by a magnetic separation; andcounting the number of oocysts.
 2. The method for measuring protozoanoocysts according to claim 1, wherein the binding factor is an antibodyagainst oocyst (hereinafter referred to as “antioocyst antibody”). 3.The method for measuring protozoan oocysts according to claim 1, whereinthe oocyst/binding factor/magnetic fine particle complexes areoocyst/antioocyst antibody/magnetic fine particle complexes formed byadding magnetic fine particles having the antioocyst antibodyimmobilized thereto to the analyte.
 4. The method for measuringprotozoan oocysts according to claim 1, wherein the binding factorcomprises the antioocyst antibody and a binding factor componentspecifically recognizing the antibody (hereinafter referred to as“antioocyst antibody-binding factor component”).
 5. The method formeasuring protozoan oocysts according to claim 1, wherein theoocyst/binding factor/magnetic fine particle complexes areoocyst/antioocyst antibody/antioocyst antibody-binding factorcomponent/magnetic fine particle complexes, which are formed by addingantibodies against oocyst to an analyte to form oocyst/antioocystantibody complexes, and subsequently adding magnetic fine particleshaving the antioocyst antibody-binding factor component against theantibody immobilized thereto.
 6. The method for measuring protozoanoocysts according to claim 1, wherein the magnetic fine particles arelabeled beforehand.
 7. The method for measuring protozoan oocystsaccording to claim 1, wherein the formed oocyst/binding factor/magneticfine particle complexes are further labeled.
 8. The method for measuringprotozoan oocysts according to claim 6, wherein the labeling is afluorescent labeling.
 9. The method for measuring protozoan oocystsaccording to claim 1, wherein the magnetic fine particles are magneticfine particles having stimuli-responsive polymers immobilized thereto.10. The method for measuring protozoan oocysts according to claim 1,wherein the protozoan is a protozoan belonging to genus Cryptosporidium.11. The method for measuring protozoan oocysts according to claim 1,wherein the analyte contains water as a solvent.
 12. A reagent fordetecting protozoan oocysts in an analyte, which comprises magnetic fineparticles of 5 to 500 nm particle diameter having antioocyst antibodiesor antioocyst antibody-binding factor components immobilized thereto.13. The reagent for detecting protozoan oocysts according to claim 12,wherein the above magnetic fine particles are magnetic fine particleshaving stimuli-responsive polymers immobilized thereto.